BCL2 expression is enriched in advanced prostate cancer with features of lineage plasticity

The widespread use of potent androgen receptor signaling inhibitors (ARSIs) has led to an increasing emergence of AR-independent castration-resistant prostate cancer (CRPC), typically driven by loss of AR expression, lineage plasticity, and transformation to prostate cancers (PCs) that exhibit phenotypes of neuroendocrine or basal-like cells. The anti-apoptotic protein BCL2 is upregulated in neuroendocrine cancers and may be a therapeutic target for this aggressive PC disease subset. There is an unmet clinical need, therefore, to clinically characterize BCL2 expression in metastatic CRPC (mCRPC), determine its association with AR expression, uncover its mechanisms of regulation, and evaluate BCL2 as a therapeutic target and/or biomarker with clinical utility. Here, using multiple PC biopsy cohorts and models, we demonstrate that BCL2 expression is enriched in AR-negative mCRPC, associating with shorter overall survival and resistance to ARSIs. Moreover, high BCL2 expression associates with lineage plasticity features and neuroendocrine marker positivity. We provide evidence that BCL2 expression is regulated by DNA methylation, associated with epithelial-mesenchymal transition, and increased by the neuronal transcription factor ASCL1. Finally, BCL2 inhibition had antitumor activity in some, but not all, BCL2-positive PC models, highlighting the need for combination strategies to enhance tumor cell apoptosis and enrich response.


Introduction
Prostate cancer (PC) is the most frequently diagnosed malerelated cancer in the Western world (1).In Europe, 1% of all men die from this disease (1,2).PC is largely driven by the androgen receptor (AR), and androgen deprivation therapy (ADT) is the cornerstone of treatment for advanced disease (3,4).However, ADT resistance invariably occurs, and tumors progress from castration-sensitive PC (CSPC) to castration-resistant PC (CRPC) (5).Metastatic CRPC (mCRPC) is a lethal malignancy with an over-all survival (OS) of 2-3 years (6,7).Although next-generation AR signaling inhibitors (ARSIs) improve OS in metastatic CSPC and CRPC, the tumors inevitably develop resistance (5).
Most commonly, treatment resistance is driven by reactivation of AR signaling (5).However, intensive selective pressure from the use of ARSIs has resulted in an increasing emergence of PCs that adapt to survive without AR signaling, often with loss of AR expression, lineage plasticity, and transformation to PCs that exhibit phenotypes of neuroendocrine or basal-like cells (5,(8)(9)(10).
The widespread use of potent androgen receptor signaling inhibitors (ARSIs) has led to an increasing emergence of ARindependent castration-resistant prostate cancer (CRPC), typically driven by loss of AR expression, lineage plasticity, and transformation to prostate cancers (PCs) that exhibit phenotypes of neuroendocrine or basal-like cells.The anti-apoptotic protein BCL2 is upregulated in neuroendocrine cancers and may be a therapeutic target for this aggressive PC disease subset.There is an unmet clinical need, therefore, to clinically characterize BCL2 expression in metastatic CRPC (mCRPC), determine its association with AR expression, uncover its mechanisms of regulation, and evaluate BCL2 as a therapeutic target and/ or biomarker with clinical utility.Here, using multiple PC biopsy cohorts and models, we demonstrate that BCL2 expression is enriched in AR-negative mCRPC, associating with shorter overall survival and resistance to ARSIs.Moreover, high BCL2 expression associates with lineage plasticity features and neuroendocrine marker positivity.We provide evidence that BCL2 expression is regulated by DNA methylation, associated with epithelial-mesenchymal transition, and increased by the neuronal transcription factor ASCL1.Finally, BCL2 inhibition had antitumor activity in some, but not all, BCL2-positive PC of 187 mCRPC biopsies with preexisting AR-NTD IHC status to investigate its association with AR-negative disease (Figure 1A and Figure 2A).These 47 biopsies were enriched for AR-negative tumors (8/47, 17%).BCL2 positivity, defined as a cytoplasmic BCL2 H score greater than 20, was observed in 28% (13/47) of the biopsies.AR-negative tumors had significantly higher cytoplasmic BCL2 immunoreactivity (Mann-Whitney U test, P < 0.001) (Figure 2B).BCL2 positivity was observed in 88% (7/8) of AR-negative tumors compared with 15% (6/40) of AR-positive tumors (Fisher's exact test, P < 0.001) (Figure 2, C and D).However, although the 6 highest BCL2-expressing tumors were AR negative, 46% (6/13) of BCL2-positive tumors were AR positive (Figure 2, B and D).There were no significant differences in BCL2 expression between biopsy sites, though the analysis was limited by small sample size (Supplemental Figure 4A).BCL2 expression was also enriched in AR-negative tumors (optical density [OD] score ≤ 0.013) in the LuCaP patient-derived xenograft (PDX) series (Mann-Whitney U test, P < 0.001) (Supplemental Figure 5,  A-D).BCL2 positivity (OD > 0.033) was observed in 71% (22/31) of AR-negative tumors and 22% (18/83) of AR-positive tumors (Fisher's exact test, P < 0.001) (Supplemental Figure 5D).This finding was further validated with transcriptomic data, revealing significantly higher BCL2 mRNA expression in AR-low mCRPC (≤ 20th percentile expression) in the Stand Up To Cancer (SU2C)/ Prostate Cancer Foundation (PCF) and SU2C/West Coast Prostate Cancer Dream Team (WCDT) cohorts (Mann-Whitney U test, P < 0.001 and P = 0.003, respectively) (Figure 2, E and F).Importantly, patients with BCL2-positive CRPC had significantly shorter OS, both from the time of CRPC diagnosis (median OS: 20.4 vs. 53.0months; HR: 2.82, 95% CI 1.43-5.56,P = 0.002) (Figure 2G) and from the time of CRPC biopsy (median OS: 8.8 vs. 13.4 months; HR: 2.03, 95% CI 1.03-4.01,P = 0.038) (Institute of Cancer Research [ICR]/Royal Marsden Hospital [RMH] CRPC IHC cohort) (Supplemental Figure 4B).
Heterogeneity of AR and BCL2 protein expression in lethal CRPC.Next, we used a rapid-autopsy CRPC cohort to investigate the degree of interpatient and intrapatient heterogeneity of AR and BCL2 expression (24).We analyzed 485 spatially separated samples from 177 mCRPC sites (58 patients).Heterogeneous expression was observed at 3 levels: interpatient, intrapatient inter-metastatic (between metastatic sites within the same patient), and intra-metastasis (within a metastatic site) (Figure 2H, Supplemental Figure 6A, and Supplemental Tables 7 and 8).The majority of the heterogeneity/variability was seen between patients (interpatient) and between metastatic sites (intrapatient inter-metastatic), but there was also a degree of intrametastasis heterogeneity (Supplemental Tables 7 and 8).In terms of metastatic site/tissue type, there was higher AR expression in bone compared with liver metastases (Supplemental Figure 7, A and B, and Supplemental Table 9).Lung metastases had higher expression of BCL2 compared with all other sites of disease, though it should be noted that the sample size was small (Supplemental Figure 7, C and D, and Supplemental Table 10).When the average expression for each metastatic site was calculated, BCL2 expression was higher in AR-negative tumors, although this did not reach statistical significance (Mann-Whitney U test, P = 0.08) (Figure 2I).However, when binary AR-NTD expression Neuroendocrine-like/basal tumors respond poorly to endocrine therapies and are associated with poor prognosis, highlighting an urgent clinical need to develop effective therapeutic strategies for this type of CRPC (11,12).
The anti-apoptotic protein BCL2 has been reported to be upregulated in various neuroendocrine cancers, including smallcell neuroendocrine CRPC (13).BCL2 promotes cell survival by sequestering pro-apoptotic proteins, including the pore-forming effector BAX, preventing mitochondrial outer membrane permeabilization (14).The BCL2 inhibitor venetoclax (ABT-199) has received FDA approval for chronic lymphocytic leukemia and acute myeloid leukemia, with clinical trials in solid tumors under way (15)(16)(17).AR activity suppresses BCL2 expression, with AR inhibition (enzalutamide) reported to upregulate BCL2 (18,19).In androgen-dependent LNCaP cells, BCL2 upregulation is required for progression to an androgen-independent state (20).Furthermore, BCL2 is upregulated in several CRPC models with primary or acquired resistance to enzalutamide (18).Thus, BCL2 targeting has been proposed as a therapeutic strategy in neuroendocrine PC, and additionally as an approach to prevent or delay resistance to ARSIs in AR-dependent disease (13,18,21).
The objectives of this study were to characterize AR expression in advanced PC, to determine its association with BCL2, to elucidate their clinical significance, and to uncover mechanisms of regulation, with the aim of exploring whether BCL2 could represent a valid therapeutic target and/or biomarker (prognostic and/or predictive).
BCL2 protein expression is enriched in AR-negative mCRPC and associates with shorter OS.BCL2 IHC was performed on 47 The molecular landscape of BCL2-positive mCRPC.To investigate the molecular landscape of BCL2-positive mCRPC, we performed gene set enrichment analysis (GSEA) using the "hallmark molecular signatures" from the Molecular Signatures Database (MSigDB, Broad Institute) for BCL2 expression in the ICR/ RMH and SU2C/PCF RNA sequencing data sets (Figure 4, A and B, Supplemental Figure 10, A and B, and Supplemental Tables 11  and 12) and mCRPC biopsy RNA sequencing data sets (23,25).In accordance with our previous findings, the "androgen response" signature was de-enriched, while the "apoptosis" signature was enriched with increasing BCL2 mRNA expression (Figure 4, A and  B).In addition, BCL2 mRNA expression was associated with "epithelial-mesenchymal transition" (EMT) and "IL-6/JAK/STAT3 signaling" (Figure 4, A and B).
To further investigate the link between EMT and BCL2, we used an LNCaP cell model with doxycycline-inducible expression of the master EMT transcription factor Snail (LNCaP-iSnail) (26).Robust Snail protein induction was observed with doxycycline treatment, which was accompanied by an upregulation of vimentin, downregulation of E-cadherin, and a shift to a mesenchymal morphology, as expected with EMT (Figure 4C and Supplemental Figure 10, C and D) (27).Induction of Snail resulted in an increase in BCL2 protein expression, while no changes were observed with the inducible GFP control (LNCaP-iGFP) (Figure 4C and Supplemental Figure 10D).In keeping with these findings, SNAI1 and VIM mRNA expression were significantly positively correlated with BCL2 expression in both mCRPC data sets (ICR/RMH r = 0.376, P <0.001, and r = 0.451, P < 0.001, respectively; SU2C/PCF r = 0.296, P < 0.001, and r = 0.386, P < 0.001, respectively) (Figure 4D).
Having shown that BCL2 expression associates with AR loss and with pathways implicated in stemness and lineage plasticity, we performed IHC for putative neuroendocrine markers (CD56, chromogranin A, and synaptophysin) on a subset of mCRPC biopsies (n = 26) (28).The 5 tumors with the highest BCL2 protein expression had concurrent expression of at least 1 neuroendocrine marker; 3 were identified as small-cell carcinomas and 2 as largecell neuroendocrine carcinomas (Figure 5, A and B, and Supplemental Table 13).Importantly, there were 4 other BCL2-positive tumors, without neuroendocrine marker expression, which were identified as adenocarcinomas.
BCL2 expression is regulated by DNA methylation and driven by the neuronal transcription factor ASCL1.To uncover further mechanisms of BCL2 expression regulation, we used whole-genome bisulfite sequencing of LuCaP PDX lines and identified a differentially methylated region (DMR) encompassing a CpG island on the BCL2 promoter.In the majority of BCL2-positive PDX models (protein OD > 0.033), the DMR was hypomethylated, and the average methylation index was lower in comparison with BCL2-negative models (P = 0.02) (Figure 6, A-C).This finding was validated in the publicly available SU2C/WCDT mCRPC patient data set, where tumors were split by BCL2 mRNA expression (< 90th percentile vs. ≥ 90th percentile) (P = 0.03) (Figure 6, D-F).
The molecular subtype of SU2C/WCDT tumors was determined using the AR, NEURO I, and NEURO II gene expression sets, as previously described (Figure 6F) (29).The transcription factor ASCL1, known to promote lineage plasticity and neuro-(OD > 0.013) was added as a population-level effect to the Bayesian generalized linear multilevel model, this association was not observed, since, although BCL2 expression was higher, the threshold for BCL2 positivity was not reached in most AR-negative tumors (Supplemental Figure 6B).
BCL2 positivity associates with resistance to ARSIs but not docetaxel, and with poor prognosis, in mCRPC.Next, we analyzed clinical data to elucidate whether BCL2 positivity associates with resistance to ARSI in CRPC.ARSI response data were available for 36 patients with BCL2 status on their CRPC biopsies (Supplemental Figure 8A).All patients received either abiraterone or enzalutamide.A ≥50% fall in prostate-specific antigen (PSA) was observed in 47.6% (10/21) of patients with BCL2-negative disease, compared with 12.5% (1/8) with BCL2 positivity (Figure 3A).Furthermore, the duration of ARSI therapy and OS from initiation of therapy were significantly shorter in patients with BCL2-positive disease (median time on therapy: 6.8 vs. 2.9 months; HR: 3.01, 95% CI 1.25-7.24,P = 0.010; median OS: 24.3 vs. 9.7 months; HR: 3.82, 95% CI 1.61-9.02,P = 0.001) (Figure 3, B and C).Interestingly, the differences in response rate, time on therapy, and OS in BCL2-positive disease were observed irrespective of AR status (Figure 3D and Supplemental Figure 8, B and C), suggesting that BCL2 positivity may denote ARSI resistance regardless of AR expression status.Docetaxel outcome data were available for 36 patients with BCL2 status on their mCRPC biopsies (Supplemental Figure 9A).There was no significant difference in time on treatment, OS from initiation of therapy, PSA response, or number of docetaxel cycles between BCL2-positive and BCL2-negative disease (Figure 3, E and F, and Supplemental Figure 9, B-G), supporting that patients with BCL2-positive mCRPC may derive more clinical benefit from docetaxel compared with ARSIs.endocrine differentiation, was highly expressed in two AR-negative/neuroendocrine-positive (AR -NE + ) tumors with concurrent high expression of BCL2 (Figure 6F) (30,31).In addition, transcriptome analysis of a cohort enriched in neuroendocrine PC showed ASCL1 exclusively expressed in tumors with low or absent AR expression, all of which also expressed BCL2 mRNA (Supplemental Figure 11) (32).
Chromatin immunoprecipitation (ChIP) studies in four AR - NE + BCL2-positive models (three LuCaP PDXs and the cell line NCI-H660) showed ASCL1 recruitment at the BCL2 locus (Figure 7A) (31).Western blot analysis of benign and PC cell lines showed that BCL2 and ASCL1 were both highly expressed in NCI-H660 (Figure 7B).Furthermore, siRNA knockdown of ASCL1 led to downregulation of BCL2 in NCI-H660 cells (Figure 7C).Taken together, these data reveal BCL2 as a transcriptional target of ASCL1 in neuroendocrine PC.
As discussed, although BCL2 expression is enriched in AR-negative mCRPC, there is a small subset of AR-positive tumors with concurrent expression of BCL2 (Figure 2, C, D, H, and I; Figure 6F; Supplemental Figure 5, A-D; and Supplemental Figure 6A).To investigate the phenotype of this subset, we interrogated transcriptomic data in an expanded SU2C/WCDT mCRPC cohort (n = 210), undertaking GSEA comparing BCL2-high/AR-high (n = 6) with BCL2-low/AR-high tumors (n = 99) (Supplemental Figure 12, A  and B).Interestingly, there was negative enrichment of the "androgen response," in keeping with the observation that BCL2 positivity associates with inferior response to ARSI and shorter OS (Supplemental Figure 12, A and B, and Supplemental Table 14).Taken together, these data support the hypothesis that BCL2 expression may denote AR independence irrespective of AR expression status.
AR and BCL2 expression in a heterogeneous mCRPC PDX model.To further elucidate the relationship of AR and BCL2 expression, we studied CP336, a PDX with 2 tumor cell populations: AR-positive/BCL2-negative and AR-negative/BCL2-positive (Figure 8,  A-C).The CP336 PDX model was established from a lymph node biopsy containing adenocarcinoma with small-cell differentiation (ICR/RMH 181) and was taken from a patient with mCRPC previously exposed to luteinizing hormone-releasing hormone (LHRH) analog, bicalutamide, and abiraterone (Figure 8, A and  B).CP336 was subsequently passaged from intact into castrated mice to generate a CP336-castrate PDX (CP336c) (Figure 8C).IHC performed on the patient biopsy and CP336/CP336c revealed that Ki67 was higher in the AR-negative/BCL2-positive tumor cell population (Figure 8, B and C).We observed predominance of the AR-negative/BCL2-positive cellular subpopulation in the CP336 model over serial passages (Figure 8C).
Targeting BCL2 in BCL2-positive PC.To investigate whether BCL2 expression associates with response to BCL2 inhibition, eight PC cell lines (one BCL2 positive and seven BCL2 negative) were treated with the BCL2 inhibitor venetoclax at various concentrations (Figure 9, A and B).Cell viability analysis revealed a substantial downward shift in the dose-response curve for the BCL2-positive NCI-H660 cell line after 72 hours of treatment compared with the BCL2-negative lines (Figure 9A).Furthermore, there was a significant increase in caspase-3/7 activity at 6 hours after 1 μM of venetoclax (unpaired, 2-tailed t test, P = 0.001) (Figure 9B).Next, three BCL2-positive PDX-derived mCRPC models -CP336c (PDX-organoid [PDX-O]), LuCaP 70CR (PDX-O), and LuCaP 136CR (primary cell culture) -were treated in vitro with venetoclax; however, there was minimal antitumor activity (Figure 9, C, E, and F).
Targeting BCL2, BCLXL, and MCL1 together triggers rapid apoptotic PC cell death.To explore functional redundancy between the 3 main anti-apoptotic BCL2 proteins (BCL2, MCL1, and BCLXL), and to enrich response, we treated CP336c PDX-Os with A-1331852 (BCLXL inhibitor; 100 nM), navitoclax (BCL2/BCLXL inhibitor; 1 μM), AZD5991 (MCL1 inhibitor; 1 μM), and navitoclax/AZD5991 combined treatment (both at 1 μM).There was marked induction of apoptosis and significant reduction in CP336c organoid viability when all 3 main anti-apoptotic BCL2 proteins were targeted (Dunnett's test, both P < 0.001) (Figure 9, E and F), suggesting that this may be an effective strategy to eliminate PC cells.This was recapitulated in 9 prostate cell lines (including benign models), though DU145 cells did not respond, as they are known to be The anti-apoptotic protein BCL2 has been proposed as an attractive therapeutic target both to prevent resistance to AR inhibition in AR-dependent PC, and to target AR-independent neuroendocrine mCRPC (13,18).The combination of enzalutamide with venetoclax has been evaluated in an early-phase clinical trial for mCRPC (ClinicalTrials.govNCT03751436), but limited antitumor activity was observed; this may be explained by the lack of molecular characterization for patient selection, including the absence of BCL2 expression analysis (36).We show that BCL2 expression is enriched in AR-negative mCRPC, in keeping with previous data from primary PC samples (37).For what we believe is the first time, we demonstrate that BCL2 positivity associates with shorter OS and with resistance to ARSI therapy in mCRPC.This finding appeared to be independent of AR expression status but was based on a small number of cases and would need to be confirmed in further studies.Importantly, there was no significant difference in outcome with docetaxel treatment, suggesting that patients with BCL2-positive mCRPC may derive more clinical benefit from docetaxel compared with ARSI.resistant to apoptosis because of loss of the pore-forming effector protein BAX (Supplemental Figure 14, A and B) (33).Thus, since such a combined approach is likely to be toxic to normal cells, targeted drug delivery technologies (e.g., antibody-drug conjugates) may be required to abrogate toxicity and induce selective cancer cell lethality (34,35).

Discussion
Targeting of the AR signaling axis is the cornerstone of treatment for advanced PC, and AR signaling remains critically important in most CRPC progression.We show that a subset of mCRPC is AR negative, associating with markedly shorter OS, highlighting the urgent clinical need to develop new effective therapies for this PC subtype.This is arguably the most extensive clinically characterized IHC study of AR in mCRPC, including 424 biopsies from 245 patients in 2 independent cohorts, as well as multiple PDX models.Given its relative rarity, there are limited clinical series reporting outcomes for aggressive PC subtypes, and none of these stratify patients by AR expression status (11,12).Although prior studies have described AR-negative mCRPC, they have not included longitudinal analyses.All the patients in our matched study had adenocarcinoma at the time of diagnosis, suggesting that loss of AR expression emerges have concurrent expression of putative neuroendocrine markers (13,28).Taken together, these data suggest that high-BCL2expressing tumors have developed an EMT or neuroendocrine/ basal phenotype.It remains to be seen whether EMT states lead to neuroendocrine/basal PC phenotypes.
It is important to note that a subset of BCL2-positive mCRPCs, ones with lower expression of BCL2, remained AR-positive/neuroendocrine marker-negative, suggesting that this process is not simply an on/off state.Despite this, these BCL2-positive/AR-positive tumors are associated with worse clinical outcomes and resistance to ARSIs.Furthermore, transcriptomic analysis revealed negative enrichment of the "androgen response" in this subset, suggesting that BCL2 positivity may denote AR independence irrespective of AR expression status.This suggests that BCL2 positivity can guide therapy decisions.However, given the small sample size, these findings should be interpreted with caution; further validation and functional studies are required.
We have also demonstrated that BCL2 mRNA expression in mCRPC associates with signaling pathways implicated in resistance to ARSI, including EMT and IL-6/JAK/STAT3 signaling (38)(39)(40)(41)(42). Recent data suggest that treatment resistance may involve increased JAK/STAT signaling and that targeting this pathway may overcome ARSI resistance (38,42).We show that BCL2 mRNA expression and SNAI1 mRNA expression are positively correlated in mCRPC, and that BCL2 protein expression is upregulated by Snail in an LNCaP cell model with doxycycline-inducible expression of Snail.Snail is a key transcription factor that drives EMT, a process that may lead to therapy resistance (43)(44)(45).In addition, in accordance with findings in neuroendocrine lung cancer, we provide evidence that BCL2 is a transcriptional target of ASCL1.ASCL1 is a transcription factor known to activate neuronal stem cell-like lineage programming and has been implicated in neuroendocrine PC (30,31,46).In keeping with this and previous studies, we show that high-BCL2-expressing tumors  supported that the BCL2-positive neuroendocrine PC cell line NCI-H660 was sensitive to venetoclax with induction of apoptosis; however, there was limited antitumor activity when BCL2-expressing PDX CRPC models were treated in vitro.However, the CP336c PDX-O, and numerous prostate cell lines, underwent rapid apoptosis when BCL2, MCL1, and BCLXL were cotargeted together, suggesting that resistance may be driven by functional redundancy between these proteins.Because of anticipated toxicity, targeting of these proteins together is unlikely to be tolerated without the use of cancer cell-specific drug delivery technologies (e.g., antibody-drug conjugates) (34,35).Another approach is to target hyperactivated oncogenic pathways that regulate the expression or activity of anti-apoptotic BCL2 proteins (e.g., MAPK or PI3K pathways), thereby specifically targeting cancer cells (47)(48)(49)(50).A further challenge is the observed AR and BCL2 hetero-Importantly, studies have shown BCL2-positive small-cell neuroendocrine cell lines to be more sensitive to BCL2 inhibition than AR-positive cell lines, with 2 of 5 (40%) CRPC small-cell neuroendocrine PDX models sensitive to navitoclax, which targets both BCLXL and BCL2 (13).Consistent with this, our studies   PDX, PDX-organoid, and cell line studies ICR/RMH patient-derived models.The CP336 (and CP336c) PDX was derived from a human CRPC lymph node biopsy using the same methods as for CP50 and CP89 (52)(53)(54).
PDX, PDX-O, primary cell culture, and cell line studies are described in Supplemental Methods.
SU2C/WCDT cohort.Whole-genome bisulfite sequencing and RNA sequencing data for the WCDT cohort were previously reported, and data were processed as described previously (57,58).Further details are described in Supplemental Methods.

Statistics
All statistical tests used are discussed in the results.Statistical tests included Wilcoxon's matched-pairs signed-rank test, Kaplan-Meier, logrank, Mann-Whitney U test, Spearman's correlation, Fisher's exact test, Dunnett's multiple-comparison test, and unpaired, 2-tailed t test and were performed using GraphPad Prism v9 or R v4.2.1.Specific details, including bioinformatic methods, can be found in Supplemental Methods.

Study approval
ICR/RMH CRPC cohort.All patients provided written informed consent and were enrolled in protocols approved by the RMH ethics review committee (reference 04/Q0801/60).
UW/FHCC CRPC IHC cohort.Samples were obtained from patients who died of mCRPC and had signed written consent for a rapid autopsy as part of the Prostate Cancer Donor Program.Tissue collection was approved by the Institutional Review Board at UW.
ICR/RMH patient-derived models.All mouse studies received approval from the ICR Animal Welfare and Ethical Review Body and were performed in accordance with the UK Animals (Scientific Procedures) Act of 1986.
UW/FHCC patient-derived models.Studies were undertaken in accordance with an approved UW Institutional Animal Care and Use Committee protocol.

Figure 1 .
Figure 1.Loss of AR protein expression occurs in a small subset of CRPC and associates with poor prognosis.(A) Summary of clinical samples analyzed for the ICR/RMH CRPC IHC patient cohort.A total of 187 mCRPC biopsies and 60 matched same-patient CSPC biopsies were analyzed.Clinical outcome data, including OS from CRPC diagnosis and CRPC biopsy date, were collected for all 187 patients.(A-D) IHC for nuclear AR-NTD was performed in 187 CRPC biopsies and 60 matched, same-patient CSPC tumors.(B) Representative micrographs for matched, same-patient samples are shown with examples of emergent loss (patient 68) and increased (patient 151) and stable (patient 169) AR-NTD protein expression.Scale bar: 50 μm.HS, H score. (C and D) Nuclear AR-NTD protein expression (H score) in 60 matched, same-patient CSPC and CRPC tumors (C) and 187 CRPC tumors (D).Medians and interquartile ranges (IQRs) are shown.Wilcoxon's matched-pairs signed-rank test was used to determine statistical significance.Red lines highlight cases of AR-NTDnegative CRPC and their matched CSPC sample.The threshold for AR negativity (H score ≤ 20) is highlighted in blue.A heterogeneous case with 2 tumor cell populations (AR positive and AR negative) is highlighted in orange and included twice.(E) Kaplan-Meier OS curves from time of CRPC diagnosis, split by AR-positive (H score > 20) and AR-negative (H score ≤ 20) tumors.HR with 95% CIs and P value for log-rank test are shown.*The heterogeneous case is included in the AR-NTD-negative group.

Figure 2 .
Figure 2. BCL2 protein expression is enriched in AR-negative disease and associates with poor prognosis.(A-D) IHC for BCL2 was performed on 47 CRPC biopsies, with preexisting AR-NTD (ICR/RMH CRPC IHC cohort).(A) Representative micrographs are shown.Scale bar: 50 μm.HS, H score. (B) Cytoplasmic BCL2 expression split by AR expression status: AR negative (AR-NTD H score ≤ 20, n = 8) and AR positive (AR-NTD H score > 20, n = 40).Median and IQRs are shown.Mann-Whitney U test was used.(C) Percentage of BCL2-positive (H score > 20) and BCL2-negative (H score ≤ 20) tumors split by AR expression status as above.Fisher's exact test was used.*The heterogeneous case with 2 tumor cell populations (AR-positive/BCL2-negative and AR-negative/BCL2-positive) is included twice (highlighted orange).(D) H scores for nuclear AR-NTD and cytoplasmic BCL2 expression.(E and F) BCL2 mRNA expression in AR-low (AR mRNA ≤20th percentile) and AR-high (AR mRNA >20th percentile) mCRPC in the SU2C/PCF (n = 210) (E) and SU2C/WCDT (n = 159) (F) cohorts.Medians and IQRs are shown.Mann-Whitney U test was used.(G) Kaplan-Meier survival curves from time of CRPC diagnosis, split by BCL2-positive and BCL2-negative tumors.HR with 95% CIs and P value for log-rank test are shown.*The heterogeneous case is included in the BCL2-positive group.(H and I) AR-NTD and BCL2 IHC was performed on 485 spatially separated samples from 177 mCRPC sites taken at rapid autopsy (58 patients).(H) Representative micrographs with examples of intrapatient inter-metastatic site heterogeneity.Scale bar: 50 μm.(I) Cytoplasmic BCL2 expression (OD) split by AR-negative (OD ≤ 0.013, n = 50) and AR-positive (OD > 0.013, n = 127) tumors.Average OD scores for all samples from each mCRPC tissue were used for this analysis.The threshold for BCL2 negativity (OD ≤ 0.033) is highlighted in blue.Median and IQRs are shown.Mann-Whitney U test was used to determine statistical significance.

Figure 3 .
Figure 3. BCL2 positivity associates with resistance to ARSI and poor prognosis in mCRPC irrespective of AR expression status, but there is no difference in response to docetaxel.(A-D) Clinical outcome data, including best PSA response, time on ARSI, and OS from starting ARSI, were collected for 36 patients.(A) Top: Waterfall plot of greatest percentage fall in PSA from baseline for 29 patients treated with either abiraterone or enzalutamide, split by BCL2 negative (blue, n = 21) and BCL2 positive (red, n = 8).*No response.Bottom: Pie charts showing percentage of patients with BCL2-negative and BCL2-positive CRPC that had a ≥50% fall in PSA from baseline.(B and C) Kaplan-Meier curves, split by BCL2-positive (H score > 20) and BCL2-negative (H score ≤ 20) tumors, showing time on ARSI (B) and OS from initiation of ARSI (C).(D) Kaplan-Meier curves, split by BCL2-positive/AR-negative, BCL2-positive/AR-positive, and BCL2-negative, for OS from initiation of ARSI.*The heterogeneous case is included in the BCL2-positive (and BCL2-positive/AR-negative) group.(E and F) Clinical outcome data, including best PSA response, time on docetaxel, number of docetaxel cycles, and OS from starting docetaxel, were collected for 36 patients.Kaplan-Meier curves, split by BCL2-positive (H score > 20) and BCL2-negative (H score ≤ 20) tumors, showing time on docetaxel (E) and OS from initiation of docetaxel (F).HR with 95% CIs and P values for the log-rank test are shown for Kaplan-Meier curves.

Figure 4 .
Figure 4. BCL2 expression associates with pathways implicated in lineage plasticity and is upregulated by Snail overexpression.(A and B) Gene set enrichment analysis (GSEA) using the "hallmark molecular signatures" was performed for BCL2 mRNA expression in the ICR/RMH (n = 95) (A) and SU2C/ PCF (n = 159) (B) CRPC RNA sequencing cohorts.Volcano plots show the false discovery rate-corrected P values (-log 10 ) against the normalized enrichment score.(C) Western blot showing protein expression of Snail, E-cadherin, BCL2, and vimentin in untreated (day 0) LNCaP-iGFP and LNCaP-iSnail, and cells treated for 5 and 7 days with doxycycline.GAPDH was used as a loading control.The experiment was performed in 2 biological replicates (see Supplemental Figure 10D).(D) Correlation between BCL2 and SNAI1 mRNA expression (left), and between BCL2 and VIM mRNA expression (right), in the ICR/RMH and SU2C/PCF data sets.Spearman's correlation was used for statistical analysis.

Figure 7 .
Figure 7. BCL2 expression is a transcriptional target of the neuronal lineage-guiding transcription factor ASCL1. (A) ASCL1 ChIP-Seq tracks in 4 AR-negative/ BCL2-positive/NE-positive PC models: 3 LuCaP PDXs and the NCI-H660 cell line.(B) Western blot showing protein expression of full-length AR (AR-FL), ASCL1, and BCL2 in benign prostate (RWPE-1 and PNT2) and PC cell lines.Vinculin and GAPDH were used as a loading control.(C) Western blot showing the impact of ASCL1 knockdown (siRNA, 72 hours, 50 nM) on BCL2 protein expression in NCI-H660 cells.Two biological replicates are shown.Vinculin was used as a loading control.

Figure 8 .
Figure 8. AR and BCL2 expression in a heterogeneous mCRPC PDX model.(A) Clinical history for patient 181, whose biopsy was used to generate CP336 PDX.CP336 PDX was developed from a lymph node biopsy containing adenocarcinoma with small-cell neuroendocrine differentiation, from a patient previously treated with agents targeting the AR signaling axis.(B) IHC for BCL2, AR-NTD, and Ki67 was performed on the lymph node biopsy used to develop CP336 PDX, revealing heterogeneous expression with 2 tumor cell populations: AR-negative/BCL2-positive and AR-positive/BCL2-negative. Representative micrographs are shown.Scale bar: 200 μm.(C) CP336-intact was passaged into castrated mice to develop CP336-castrate (CP336c).IHC was performed at different time points.Representative micrographs are shown.Scale bar: 100 μm.